Vitamin D is a steroid hormone which is involved in the human body's system for regulating calcium by influencing the intestinal absorption of calcium and renal re-absorption of calcium. Vitamin D is a fat-soluble hormone that the body can synthesize. There are two major types of vitamin D: vitamin D2 (ergocalciferol), which is synthesized by plants and is not produced by the human body; and vitamin D3 (cholecalciferol), which is made on the human body's skin when exposed to sunlight. Vitamin D binds to two proteins in the bloodstream, albumin and vitamin D binding protein. Once in the bloodstream, the bound vitamin D is transported to the liver, wherein it is transformed into 25-hydroxyvitamin D or calcidiol, which is the primary circulating form of vitamin D and the most commonly measured form in serum. In the kidneys, it is transformed into 1,25 dihydroxy vitamin D or calcitriol, which is the biologically active form of vitamin D.
Vitamin D deficiency is a worldwide problem. It is estimated that over 1 billion people worldwide are either vitamin D deficient or insufficient, including over 40% of elderly US population. Vitamin D is essential for maintaining strong bones because it helps the body use calcium from the diet. Traditionally, vitamin D deficiency has been associated with rickets, a disease in which the bone tissue doesn't properly mineralize, leading to soft bones and skeletal deformities. But increasingly, research indicates the importance of vitamin D in protecting against a number of other health problems. Vitamin D deficiency is associated with hypocalcemia—the presence of low serum calcium levels in the blood, and hypophosphatemia—an abnormally low level of phosphate in the blood, and elevated alkaline phosphatase. Too little vitamin D can pose health risks. Low blood levels of the vitamin have been associated with increased risk of death from cardiovascular disease, cognitive impairment in older adults, severe asthma in children, and cancer. In addition, insufficient intake or production of vitamin D can be caused by a decreased absorption or excessive loss in the gastrointestinal tract, increased vitamin D metabolism, or impaired conversion of vitamin D to 25-hydroxy vitamin D.
Even though 25-hydroxyvitamin D is the preferred vitamin D analyte, measuring it is not straightforward. In serum, the hormone is completely bound to proteins. Furthermore, the normal protein levels in human serum are capable of binding hundreds of nanograms of vitamin D. To measure vitamin D in blood, it must first be released from its binding protein. Analysis methods for vitamin D typically employ LC-MS/MS, liquid chromatography-tandem mass spectrometry analysis protocols. In such protocols the binding protein is precipitated from serum sample. Following the precipitation of the binding protein, the vitamin D is extracted from the sample and then an analysis of organic material is performed. Analytical methods that can accurately and quantitatively identify both forms of vitamin D and their metabolites are essential for diagnosis of vitamin D-related disorders, and for monitoring therapeutic response in patients being treated for vitamin D deficiency.
In pharmaceutical bioanalysis, researchers develop and run various assays to quantitate drugs, pharmaceutical candidates, and their metabolites in biological fluids, such as serum and plasma. The data resulting from these assays are used to help determine the pharmacodynamic and pharmacokinetic properties as well as the toxic and therapeutic concentrations of existing and emerging pharmaceutical compounds in living cells, tissues, and animals. Bioanalytical evaluation is a critical element of the analytical information utilized during the course of drug development, including the pre-clinical stage, the clinical stage, and the therapeutic drug monitoring stage.
Although advances in Liquid Chromatography-Mass Spectrometry (LC-MS) technology have provided improvements in analytical techniques by increasing throughput and improving sensitivity, sample preparation continues to be a critical component of bioanalysis.
The objectives of sample preparation are: 1) concentrating the analyte or analytes of interest; 2) removing interfering compositions which are present in the original sample; and, 3) altering the sample environment to one which is more compatible with the analytical system. Thus the selection of sample preparation techniques range from simple dilution to more elaborate techniques, such as dialysis, ultrafiltration, supercritical fluid extraction, liquid-liquid extraction, and monolithic chromatography. The most widely used techniques in pharmaceutical bioanalysis are protein precipitation, liquid-liquid extraction (LLE), and solid phase extraction (SPE).
Typically, protein precipitation involves dilution of biological samples (e.g. plasma) with a protein precipitating reagent, such as acetonitrile, typically at a volume ratio between 1:3 and 1:4. The diluted sample is vortexed, and the resulting precipitated proteins are removed using filtration or centrifugation methods. The filtrate or supernatant is analyzed without further processing by LC-MS or LC-MS-MS systems. LC-MS-MS, or liquid chromatography-mass spectrometry-mass spectrometry, is an analytical technique in which an additional mass spectrometry is performed on a fragmented ion selected from the first mass spectrometry, and is most often used to sequence peptides. Protein precipitation is relatively simple, and does not require separate procedures for different types of bioanalytical samples. Furthermore, protein precipitation is amenable to high throughput applications which employ automated or computer-directed liquid handlers using multi-well technology. However, there are some major drawbacks to the protein precipitation technique.
Currently, many clinical diagnostic labs still use traditional immunoassays which cannot differentiate between different forms of metabolite, like 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3. LC-MS/MS (liquid chromatography-tandem mass spectrometry) instruments enable a more selective and high sensitive detection method. However, there are problems related to this new technology. Vitamin D metabolites are strongly bonded to proteins in human plasma or serum. Ordinary solid phase extraction or liquid-liquid extract method cannot break this bonding, and therefore cannot completely extract vitamin D metabolites from plasma or serum, directly. Current analytical methods typically require two steps: firstly organic solvents are employed to break the protein bonding, and secondly, the free analytes are further extracted either by solid phase extraction or liquid-liquid extraction techniques. These two step procedures require a lot of sample transferring and labeling. The procedures are time consuming, and these methods cannot be adapted to high volume methods.
Typically, these solid phase extraction (SPE) based methods typically do not completely remove all of the phospholipid compounds, the final results may not be consistent. All vitamin D metabolites and some phospholipid compounds are relatively hydrophobic compounds, and most reversed phase SPE sorbents systems for extraction of the vitamin D metabolites separate the various compounds by hydrophobic interactions. Thus, vitamin D metabolites, especially 25-hydroxy vitamin D, coelute with hydrophobic phospholipids and produce erroneous results. Other methods which employ liquid-liquid extraction to precipitate the protein from the bound vitamin D metabolites are time consuming are subject to handling errors and require frequent maintenance of mass spectrometer equipment to provide consistent results.
US Patent Publication 20080213906 to Supelco discloses a method and device for the preparation of biological samples for subsequent LC-MS analysis using a combined and concurrent protein precipitation and solid phase extraction (SPE) process. The method uses an integrated combination of protein precipitation, filtration, and SPE using a zirconia-coated chromatographic media. According to the method, interfering compounds, such as proteins and phosphate-containing compounds, are eliminated from the biological samples, providing a higher degree of analyte response during LC-MS analysis. The protocol requires adding formic acid to the reagent. According to the method, an organic acid, such as formic acid is required to remove the phosphate compounds.
US Publication No. 2013/0053588 discloses methods, kits and devices for separating phospholipids and proteins from small molecules in biochemical samples. The apparatus includes a wetting barrier, at least one frit and a separation media. The wetting barrier is adapted to 1) retain the liquid sample and a protein precipitating agent in the sample receiving area under a first force, thereby facilitating the formation of a protein precipitate and a processed sample, and 2) flow the processed sample through the wetting barrier and separation media under a second force, wherein the second force is greater than the first force, thereby retaining the protein precipitate in the sample receiving area, retaining phospholipids in the separation media, and eluting small molecules.
Improved methods are sought to free vitamin D metabolites from binding proteins, remove the protein and selectively bind phospholipids so that metabolites of vitamin D can be extracted from serum samples in a single step protocol.